Abstract Antimicrobial Peptides Research Articles

Hermetia illucens as a Source of Antimicrobial Peptides – A Review of in vitro and in vivo Studies

Abstract Antimicrobial peptides (AMPs) are the focus of this manuscript, as bioconservative molecules that constitute a major defense mechanism in many organisms. Various antimicrobial peptides have been isolated and identified, but AMPs derived from Hermetia illucens (HI) will be the focus of this review. The review focuses mainly on cecropins, defensins, and attacins. Hermetia illucens is a remarkable organism adapted to life in a diverse, often highly polluted environment, and its resilience is largely attributed to amps. Amps are active against many bacterial and fungal species and also act to induce the osmotic lysis of protozoa. They attack pathogenic microorganisms without damaging host cells in the process. Given the increasing antibiotic resistance of many bacterial strains in animal production, amps appear to be a tempting alternative as substances that limit and inhibit the growth and development of pathogens, as well as an option in veterinary medicine as potential substitutes for antibiotics, the administration of which must be limited in the european union. The amp content of HI larvae extracts, which determines their activity, depends on the larval diet and the solvent used. This review presents in vitro studies on the effects of amps from Hermetia illucens on microorganisms and in vivo studies on the potential of Hi larvae meal as a feed supplement.

IAMPCN: a deep-learning approach for identifying antimicrobial peptides and their functional activities.

Antimicrobial peptides (AMPs) are short peptides that play crucial roles in diverse biological processes and have various functional activities against target organisms. Due to the abuse of chemical antibiotics and microbial pathogens' increasing resistance to antibiotics, AMPs have the potential to be alternatives to antibiotics. As such, the identification of AMPs has become a widely discussed topic. A variety of computational approaches have been developed to identify AMPs based on machine learning algorithms. However, most of them are not capable of predicting the functional activities of AMPs, and those predictors that can specify activities only focus on a few of them. In this study, we first surveyed 10 predictors that can identify AMPs and their functional activities in terms of the features they employed and the algorithms they utilized. Then, we constructed comprehensive AMP datasets and proposed a new deep learning-based framework, iAMPCN (identification of AMPs based on CNNs), to identify AMPs and their related 22 functional activities. Our experiments demonstrate that iAMPCN significantly improved the prediction performance of AMPs and their corresponding functional activities based on four types of sequence features. Benchmarking experiments on the independent test datasets showed that iAMPCN outperformed a number of state-of-the-art approaches for predicting AMPs and their functional activities. Furthermore, we analyzed the amino acid preferences of different AMP activities and evaluated the model on datasets of varying sequence redundancy thresholds. To facilitate the community-wide identification of AMPs and their corresponding functional types, we have made the source codes of iAMPCN publicly available at https://github.com/joy50706/iAMPCN/tree/master. We anticipate that iAMPCN can be explored as a valuable tool for identifying potential AMPs with specific functional activities for further experimental validation.

Antimicrobial peptides do not directly contribute to aging in Drosophila, but improve lifespan by preventing dysbiosis.

Antimicrobial peptides (AMPs) are innate immune effectors first studied for their role in host defence. Recent studies have implicated these peptides in the clearance of aberrant cells and in neurodegenerative syndromes. In Drosophila, many AMPs are produced downstream of Toll and Imd NF-κB pathways upon infection. Upon aging, AMPs are upregulated, drawing attention to these molecules as possible causes of age-associated inflammatory diseases. However, functional studies overexpressing or silencing these genes have been inconclusive. Using an isogenic set of AMP gene deletions, we investigated the net impact of AMPs on aging. Overall, we found no major effect of individual AMPs on lifespan, with the possible exception of Defensin. However, ΔAMP14 flies lacking seven AMP gene families displayed reduced lifespan. Increased bacterial load in the food of aged ΔAMP14 flies suggested that their lifespan reduction was due to microbiome dysbiosis, consistent with a previous study. Moreover, germ-free conditions extended the lifespan of ΔAMP14 flies. Overall, our results did not point to an overt role of individual AMPs in lifespan. Instead, we found that AMPs collectively impact lifespan by preventing dysbiosis during aging.

Handcrafted versus non-handcrafted (self-supervised) features for the classification of antimicrobial peptides: complementary or redundant?

Antimicrobial peptides (AMPs) have received a great deal of attention given their potential to become a plausible option to fight multi-drug resistant bacteria as well as other pathogens. Quantitative sequence-activity models (QSAMs) have been helpful to discover new AMPs because they allow to explore a large universe of peptide sequences and help reduce the number of wet lab experiments. A main aspect in the building of QSAMs based on shallow learning is to determine an optimal set of protein descriptors (features) required to discriminate between sequences with different antimicrobial activities. These features are generally handcrafted from peptide sequence datasets that are labeled with specific antimicrobial activities. However, recent developments have shown that unsupervised approaches can be used to determine features that outperform human-engineered (handcrafted) features. Thus, knowing which of these two approaches contribute to a better classification of AMPs, it is a fundamental question in order to design more accurate models. Here, we present a systematic and rigorous study to compare both types of features. Experimental outcomes show that non-handcrafted features lead to achieve better performances than handcrafted features. However, the experiments also prove that an improvement in performance is achieved when both types of features are merged. A relevance analysis reveals that non-handcrafted features have higher information content than handcrafted features, while an interaction-based importance analysis reveals that handcrafted features are more important. These findings suggest that there is complementarity between both types of features. Comparisons regarding state-of-the-art deep models show that shallow models yield better performances both when fed with non-handcrafted features alone and when fed with non-handcrafted and handcrafted features together.

An Efficient Evaluation System Accelerates α-Helical Antimicrobial Peptide Discovery and Its Application to Global Human Genome Mining.

Antimicrobial peptides (AMPs), as an important part of the innate immune system of an organism, is a kind of promising drug candidate for novel antibiotics due to their unique antibacterial mechanism. However, the discovery of novel AMPs is facing a great challenge due to the complexity of systematic experiments and the poor predictability of antimicrobial activity. Here, a novel and comprehensive screening system, the Multiple Descriptor Multiple Strategy (MultiDS), was proposed based on 59 physicochemical and structural parameters, three strategies, and four algorithms for the mining of α-helical AMPs. This approach was applied to mine the encrypted peptide antibiotics from the global human genome, including introns and exons. A library of approximately 70 billion peptides with 15–25 amino acid residues was screened by the MultiDS system and generated a list of peptides with the Multiple Descriptor Index (MD index) scores, which was the core part of the MultiDS system. Sixty peptides with top MD scores were chemically synthesized and experimentally tested their antimicrobial activity against 10 kinds of Gram-positive bacteria, Gram-negative bacteria (including drug-resistant pathogens). A total of fifty-nine out of 60 (98.3%) peptides exhibited antimicrobial activity (MIC ≤ 64 μg/mL), and 24 out of 60 (40%) peptides showed high activity (MIC ≤ 2 μg/mL), validating the MultiDS system was an effective and predictive screening tool with high hit rate and superior antimicrobial activity. For further investigation, AMPs S1, S2, and S3 with the highest MD scores were used to treat the skin infection mouse models in vivo caused by Escherichia coli, drug-resistance Escherichia coli, and Staphylococcus aureus, respectively. All of S1, S2, and S3 showed comparable therapeutic effects on promoting infection healing to or even better than the positive drug levofloxacin. A mechanism study discovered that rapid bactericidal action was caused by cell membrane disruption and content leakage. The MultiDS system not only provides a high-throughput approach that allows for the mining of candidate AMPs from the global genome sequence but also opens up a new route to accelerate the discovery of peptide antibiotics.

Whole-Genome Sequencing and Comparative Genomic Analysis of Antimicrobial Producing Streptococcus lutetiensis from the Rumen.

Antimicrobial peptides (AMPs) can efficiently control different microbial pathogens and show the potential to be applied in clinical practice and livestock production. In this work, the aim was to isolate AMP-producing ruminal streptococci and to characterize their genetic features through whole-genome sequencing. We cultured 463 bacterial isolates from the rumen of Nelore bulls, 81 of which were phenotypically classified as being Streptococcaceae. Five isolates with broad-range activity were genome sequenced and confirmed as being Streptococcus lutetiensis. The genetic features linked to their antimicrobial activity or adaptation to the rumen environment were characterized through comparative genomics. The genome of S. lutetiensis UFV80 harbored a putative CRISPR-Cas9 system (Type IIA). Computational tools were used to discover novel biosynthetic clusters linked to the production of bacteriocins. All bacterial genomes harbored genetic clusters related to the biosynthesis of class I and class II bacteriocins. SDS-PAGE confirmed the results obtained in silico and demonstrated that the class II bacteriocins predicted in the genomes of three S. lutetiensis strains had identical molecular mass (5197 Da). These results demonstrate that ruminal bacteria of the Streptococcus bovis/equinus complex represent a promising source of novel antimicrobial peptides.

Nanoencapsulated Lippia rotundifolia antimicrobial peptide: synthesis, characterization, antimicrobial activity, and cytotoxicity evaluations.

Antimicrobial peptides (AMP) are promising novel antibiotics but exhibit low stability and can be toxic. The AMP encapsulation can be used to protect the drug and control its release rates. The Lr-AMP1f encapsulated into chitosan nanoparticle (NP) by ionic gelation method reached 90% efficiency. The results indicated that the hydrodynamic particle size of NPs increased from 196.1 ± 3.14nm (free NP) to 228.1 ± 12.22nm (nanoencapsulated Lr-AMP1f), while the atomic force microscope showed the spherical shape. The Zeta potential of the nanoencapsulated Lr-AMP1f was high (+ 35mV). These AMP-loaded NPs exhibited stability for up to 21days of storage. The minimum inhibitory concentration (MIC) of free Lr-AMP1f was 8µg/mL for E. coli and S. epidermidis. However, the nanoencapsulated Lr-AMP1f produced a bacteriostatic effect against both bacteria at 8µg/mL. The MIC of nanoencapsulated Lr-AMP1f was 16µg/mL for E. coli and 32 for S. epidermidis. Nanoencapsulated Lr-AMP1f was nontoxic to HEK293 cells. Promisingly, chitosan NP can be used as a vehicle for the antibacterial application of new AMP (Lr-AMP1f).

The lipid components of high-density lipoproteins (HDL) are essential for the binding and transportation of antimicrobial peptides in human serum

Antimicrobial peptides (AMPs) have been developed for the treatment of bacterial infections, but their applications are limited to topical infections since they are sequestered and inhibited in serum. Here we have discovered that the inhibition of AMPs by human serum was mediated through high-density lipoproteins (HDL) which are known to remove cholesterol from peripheral tissues. The susceptibility of AMPs to HDL varied depending on the degree of hydrophobicity of AMPs and their binding affinities to HDL. The phospholipids, such as phosphatidylcholine, of HDL were essential for AMP-binding. The dynamic binding interactions between AMPs and HDL were mediated through the hydrophobic interactions rather than by ionic strength. Interestingly, some AMPs, such as SMAP29, dissociated from the AMP-HDL complex and translocated to bacteria upon contact, while some AMPs, such as LL37, remained in complex with HDL. These results suggest that HDL binds AMPs and facilitates the translocation of them to the bacteria.

In Vivo Evaluation of ECP Peptide Analogues for the Treatment of Acinetobacter baumannii Infection.

Antimicrobial peptides (AMPs) are alternative therapeutics to traditional antibiotics against bacterial resistance. Our previous work identified an antimicrobial region at the N-terminus of the eosinophil cationic protein (ECP). Following structure-based analysis, a 30mer peptide (ECPep-L) was designed that combines antimicrobial action against Gram-negative species with lipopolysaccharides (LPS) binding and endotoxin-neutralization activities. Next, analogues that contain non-natural amino acids were designed to increase serum stability. Here, two analogues were selected for in vivo assays: the all-D version (ECPep-D) and the Arg to Orn version that incorporates a D-amino acid at position 2 (ECPep-2D-Orn). The peptide analogues retained high LPS-binding and anti-endotoxin activities. The peptides efficacy was tested in a murine acute infection model of Acinetobacter baumannii. Results highlighted a survival rate above 70% following a 3-day supervision with a single administration of ECPep-D. Moreover, in both ECPep-D and ECPep-2D-Orn peptide-treated groups, clinical symptoms improved significantly and the tissue infection was reduced to equivalent levels to mice treated with colistin, used as a last resort in the clinics. Moreover, treatment drastically reduced serum levels of TNF-α inflammation marker within the first 8 h. The present results support ECP-derived peptides as alternative candidates for the treatment of acute infections caused by Gram-negative bacteria.

Evolution-Based Protein Engineering for Antifungal Peptide Improvement.

Antimicrobial peptides (AMPs) have been considered as the alternatives to antibiotics because of their less susceptibility to microbial resistance. However, compared with conventional antibiotics they show relatively low activity and the consequent high cost and nonspecific cytotoxicity, hindering their clinical application. What’s more, engineering of AMPs is a great challenge due to the inherent complexity in their sequence, structure, and function relationships. Here, we report an evolution-based strategy for improving the antifungal activity of a nematode-sourced defensin (Cremycin-5). This strategy utilizes a sequence-activity comparison between Cremycin-5 and its functionally diverged paralogs to identify sites associated with antifungal activity for screening of enhanceable activity-modulating sites for subsequent saturation mutagenesis. Using this strategy, we identified a site (Glu-15) whose mutations with nearly all other types of amino acids resulted in a universally enhanced activity against multiple fungal species, which is thereby defined as a Universally Enhanceable Activity-Modulating Site (UEAMS). Especially, Glu15Lys even exhibited >9-fold increased fungicidal potency against several clinical isolates of Candida albicans through inhibiting cytokinesis. This mutant showed high thermal and serum stability and quicker killing kinetics than clotrimazole without detectable hemolysis. Molecular dynamic simulations suggest that the mutations at the UEAMS likely limit the conformational flexibility of a distant functional residue via allostery, enabling a better peptide–fungus interaction. Further sequence, structural, and mutational analyses of the Cremycin-5 ortholog uncover an epistatic interaction between the UEAMS and another site that may constrain its evolution. Our work lights one new road to success of engineering AMP drug leads.

BING, a novel antimicrobial peptide isolated from Japanese medaka plasma, targets bacterial envelope stress response by suppressing cpxR expression

Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.

β-Defensin from the Asian Sea Bass, Lates calcarifer: Molecular Prediction and Phylogenetic Analysis

Antimicrobial peptides (AMPs) are an important element of the innate immune system of all living organisms and serve as a barrier that safeguards the organisms against a wide range of pathogens. Fishes are proven to be a prospective source of AMPs, and β-defensins form an important family of AMPs with potent antimicrobial, chemotactic and immunomodulatory activities. The present study reports a β-defensin AMP sequence (Lc-BD) from the Asian sea bass, Lates calcarifer, a commercially important fish species in tropical and subtropical regions of Asia and the Pacific. A 202-bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) was obtained from the mRNA of gill tissue by RT-PCR. The deduced aa sequence of Lc-BD possessed a signal and a mature peptide region with 20 and 43 aa residues, respectively. Lc-BD was characterized at the molecular level, and a molecular weight of 5.24 kDa and a net charge of +4.5 was predicted for the mature peptide. The molecular characterization of Lc-BD revealed the presence of three intramolecular disulphide bonds involving the sixconserved cysteine residues in the sequence, and the phylogenetic analysis of Lc-BD showed a close relationship with β-defensins from fishes like Siniperca chuatsi, Argyrosomus regius, Trachinotus ovatus and Oplegnathus fasciatus.

PCL-1, a Trypsin-Resistant Peptide, Exerts Potent Activity Against Drug-Resistant Bacteria.

Antimicrobial peptides (AMPs), which hold tremendous promise in overcoming the emergence of drug resistance, are limited in wide clinical applications due to their instability, especially against trypsin. Herein, we designed six peptide mutants based on the cathelicidin CATHPb2, followed by screening. Pb2-1, which showed the best activity against drug-resistant bacteria among these mutants, was selected to be combined with the trypsin inhibitory loop ORB-C to obtain two hybrid peptides: PCL-1 and Pb2-1TI. Notably, both of the hybrid peptides exhibited a remarkable enhancement in trypsin resistance compared with Pb2-1. The tests showed that PCL-1 displayed broad-spectrum antimicrobial activity that was superior to that of Pb2-1TI. In addition, PCL-1 had relatively lower cytotoxicity than Pb2-1TI towards the L02 and HaCaT cell lines and negligible hemolysis, as well as tolerance to high concentrations of salt, extreme pH, and temperature variations. In vivo, PCL-1 effectively improved the survival rate of mice that were systemically infected with drug-resistant Escherichia coli through efficient bacterial clearance from the blood and organs. With regard to mode of action, PCL-1 damaged the integrity of the bacterial cell membrane and attached to the membrane surface while bound to bacterial genomic DNA to eventually kill the bacteria. Altogether, the trypsin-resistant peptide PCL-1 is expected to be a candidate for the clinical treatment of bacterial infections.

Molecular docking analysis of antimicrobial peptides with the CXCL1 protein target for colorectal cancer

Antimicrobial peptides (AMPs) play a prominent role in drug discovery due to the rapid increase in drug resistant infections. Hence, we report the molecular docking analysis of antimicrobial peptides MREEKKERKRD and MVQGAKRGGRLHRV with the target protein CXCL1 in the context of colorectal cancer for further consideration in drug discovery.

Molecular docking analysis of antimicrobial peptides with the CXCL1 protein target for colorectal cancer.

Antimicrobial peptides (AMPs) play a prominent role in drug discovery due to the rapid increase in drug resistant infections. Hence, we report the molecular docking analysis of antimicrobial peptides MREEKKERKRD and MVQGAKRGGRLHRV with the target protein CXCL1 in the context of colorectal cancer for further consideration in drug discovery.

Comprehensive assessment of machine learning-based methods for predicting antimicrobial peptides.

Antimicrobial peptides (AMPs) are a unique and diverse group of molecules that play a crucial role in a myriad of biological processes and cellular functions. AMP-related studies have become increasingly popular in recent years due to antimicrobial resistance, which is becoming an emerging global concern. Systematic experimental identification of AMPs faces many difficulties due to the limitations of current methods. Given its significance, more than 30 computational methods have been developed for accurate prediction of AMPs. These approaches show high diversity in their data set size, data quality, core algorithms, feature extraction, feature selection techniques and evaluation strategies. Here, we provide a comprehensive survey on a variety of current approaches for AMP identification and point at the differences between these methods. In addition, we evaluate the predictive performance of the surveyed tools based on an independent test data set containing 1536 AMPs and 1536 non-AMPs. Furthermore, we construct six validation data sets based on six different common AMP databases and compare different computational methods based on these data sets. The results indicate that amPEPpy achieves the best predictive performance and outperforms the other compared methods. As the predictive performances are affected by the different data sets used by different methods, we additionally perform the 5-fold cross-validation test to benchmark different traditional machine learning methods on the same data set. These cross-validation results indicate that random forest, support vector machine and eXtreme Gradient Boosting achieve comparatively better performances than other machine learning methods and are often the algorithms of choice of multiple AMP prediction tools.

Synthetic antimicrobial agents inhibit aflatoxin production.

Antimicrobial peptides (AMPs) are biologically active molecules that can eradicate bacteria by destroying the bacterial membrane structure, causing the bacteria to rupture. However, little is known about the extent and effect of AMPs on filamentous fungi. In this study, we synthesized small molecular polypeptides by an inexpensive heat conjugation approach and examined their effects on the growth of Aspergillus flavus and its secondary metabolism. The antimicrobial agents significantly inhibited aflatoxin production, conidiation, and sclerotia formation in A. flavus. Furthermore, we found that the expression of aflatoxin structural genes was significantly inhibited, and the intracellular reactive oxygen species (ROS) level was reduced. Additionally, the antimicrobial agents can change membrane permeability. Overall, our results demonstrated that antimicrobial agents, safe to mammalian cells, have an obvious impact on aflatoxin production, which indicated that antimicrobial agents may be adopted as a new generation of potential agents for controlling aflatoxin contamination.

A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide–lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising d-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

Antimicrobial peptides in the gut-brain axis: A straightforward review to unravel some missing links.

Antimicrobial peptides (AMPs) are intriguing molecules, able to directly kill several microorganisms and to regulate multiple aspects of the immune response. Despite the extensive studies on the role of AMPs in the epithelial barrier, placing them as a pivotal line of defense against pathogen invasion, little attention has been directed to their role in the maintenance and modulation of the gut microbiota and, by consequence, of the homeostasis of extra intestinal tissues. Here, we review the recent literature about the microbiome-gut-brain axis, focusing on the role of AMPs in this scenario. We provide a straightforward revision of current data in order to provide an overview of the subject, discussing more in depth some points that, in our opinion, are crucial and have received little attention.

Inhibition of the ATP synthase sensitizes Staphylococcus aureus towards human antimicrobial peptides

Antimicrobial peptides (AMPs) are an important part of the human innate immune system for protection against bacterial infections, however the AMPs display varying degrees of activity against Staphylococcus aureus. Previously, we showed that inactivation of the ATP synthase sensitizes S. aureus towards the AMP antibiotic class of polymyxins. Here we wondered if the ATP synthase similarly is needed for tolerance towards various human AMPs, including human β-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the WT, even when the oxidative burst is inhibited.These results show that the staphylococcal ATP synthase enhance tolerance of S. aureus towards some human AMPs and this indicates that inhibition of the ATP synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMPs of the innate immune system.